26 GHELARDINI ET AL.
© 1997 Wiley-Liss, Inc.
DRUG DEVELOPMENT RESEARCH 42:26–34 (1997)
Research Article
Central Muscarinic Antinociception Induced by ET-142
and SS-20 in Rodents
Carla Ghelardini,1* Nicoletta Galeotti,1 Lia Fantetti,1 Fulvio Gualtieri,2 Silvia Dei,2 Serena Scapecchi,2 and Alessandro Bartolini1
1
Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy
2
Department of Pharmaceutical Sciences, University of Florence, Florence, Italy
The antinociceptive effects of ET-142 (10–50 mg kg–1 sc; 10–30 µg per mouse icv) and SS-20 (10–50 mg kg–1 sc; 5–30 µg per mouse icv) were examined in mice by using the hot-plate and abdominal constriction tests. A similar antinociceptive profile for both compounds (20–40 mg kg–1 ip) was also observed in rats using the paw pressure test. In the antinociceptive dose-range, ET-142 and SS-20 did not impair mouse gross behavior and motor coordination evaluated, respectively, by the Irwin and rotarod tests. The increase in the pain threshold produced by ET-142 and SS-20 was prevented by atropine, dicyclomine, pirenzepine, and hemicholinium-3, but not by naloxone, atropine methyl bromide, and CGP 35348. In vitro experiments showed that the two investigated compounds amplified electrically evoked guinea pig ileum contractions. On the basis of the above data, it can be postulated that ET-142 and SS-20 exert their antinociceptive effect through a potentiation of central cholinergic transmission. Drug Dev. Res. 42:26–34, 1997. © 1997 Wiley-Liss, Inc.
Key words: analgesia; antinociception; cholinergic system
Strategy, Management and Health Policy Venture Capital Enabling Technology Preclinical Research Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics Clinical Development Phases I-III Regulatory, Quality, Manufacturing Postmarketing Phase IV
Contract grant sponsor: Fidia S.p.A., Abano Terme, Italy; Con-tract grant sponsor: Ministero dell’Università e della Ricerca Scientifica e Tecnologia, MURST.
*Correspondence to: Dr. Carla Ghelardini, Department of Pharmacology, Viale G.B. Morgani 65, I-50134 Florence, Italy; E-mail: bartolin@stat.ds.unifi.it.
Received 15 July 1997; accepted 12 August 1997.
INTRODUCTION
It has long been known that acetylcholine (ACh) [Pedigo et al., 1975], selective M1 agonists such as
McN-A-343 and AF-102B [Bartolini et al., 1992], nonselective muscarinic agonists like tremorine [Lenke, 1958], ox-otremorine [George et al., 1962; Bartolini et al., 1987], arecoline [Herz, 1962], pilocarpine [Hendershot and Forsaith, 1959], and cholinesterase inhibitors such as phy-sostigmine [Harris et al., 1969; Ireson, 1970] and diisopropyl fluorophosphate [Lentz et al., 1969] induce antinociception in laboratory animals by activating the cholinergic system. It has also been reported that the antimuscarinic drug atropine, at very low doses, was able to induce a central cholinergic antinociception in labo-ratory animals regardless of the route of administration and the noxious stimulus applied [Ghelardini et al., 1990]. Interestingly, the typical cholinergic symptomatology did not accompany this antinociceptive activity. The atropine-induced increase in the pain threshold was attributable
to the R-(+)-enantiomer of atropine, R-(+)-hyoscyamine, since S-(–)-hyoscyamine was ineffective in all anti-nociceptive tests used [Ghelardini et al., 1992]. Investi-gating the antinociceptive effect of atropine using microdialysis techniques, it was demonstrated that R-(+)-hyoscyamine, at effective doses, produced an increase in ACh release from rat cerebral cortex in vivo [Ghelardini et al., 1997].
In order to obtain new cholinergic modulators, the compounds labeled ET-142 (2-(4-chlorophenoxy)-propionic acid, endo
yl ester) and SS-20 (2-[(4-chlorophenyl)thio]propionic acid, endo 8-methyl-8-azabicyclo[3.2.1]oct-3-yl ester) (Fig. 1), structurally related to atropine, have been syn-thesized [Gualtieri et al., 1994] and their potential antinociceptive properties were investigated in mice and rats.
MATERIALS AND METHODS Animals
Male Swiss albino mice (23–30 g) and rats (200– 300 g) from Morini (San Polo d’Enza, Italy) and guinea pigs (150–200 g) from Rodentia (Bergamo, Italy) breed-ing farms were used. Fifteen mice, four rats, and four guinea pigs were housed per cage. The cages were placed in the experimental room 24 h before the test for accli-matization. The animals were kept at 23 ± 1°C with a 12 h light/dark cycle, light at 7 AM, with food and water ad
libitum. All experiments were carried out according to the guidelines of the European Community Council.
Hot Plate Test
The method adopted has been described by O’Callaghan and Holtzman [1975]. Mice were placed inside a stainless steel container, thermostatically set at 52.5 ± 0.1°C in a precision water-bath from KW
Me-chanical Workshop (Siena, Italy). Reaction times (sec), were measured with a stop-watch before and at regular intervals up to a maximum of 45 min after treatment. The endpoint used was the licking of the fore or hind paws. Mice with a licking latency below 12 and over 18 sec in the pretest were rejected (30%). An arbitrary cutoff time of 45 sec was adopted.
Abdominal Constriction Test
Mice were injected ip with a 0.6% solution of ace-tic acid (10 ml kg–1), according to Koster et al. [1959]. The number of stretching movements was counted for 10 min, starting 5 min after acetic acid injection.
Paw Pressure Test
The nociceptive threshold in the rat was determined with an analgesimeter (Ugo Basile, Varese, Italy), accord-ing to the method described by Leighton et al. [1988]. Threshold pressure was measured before treatment and 15, 30, and 45 min after treatment. Rats scoring below 30 g or over 85 g in the test prior to drug administration were rejected (25%). An arbitrary cutoff value of 250 g was adopted.
Rotarod Test
The apparatus consisted of a base platform and a rotating rod of 3 cm diameter with a non-slippery sur-face. This rod was placed at a height of 15 cm from the base. The rod, 30 cm in length, was divided into five equal sections by six disks. Thus, up to five mice were tested simultaneously on the apparatus, with a rod-rotating speed of 16 rpm. The integrity of motor coordination was assessed on the basis of endurance time of the animals on the rotating rod, expressed in seconds, according to Kuribara et al. [1977]. One day before the test the ani-mals were trained twice. On the day of the test only the mice that were able to stay balanced on the rotating rod between 70 and 120 sec (cutoff time) were selected for testing. The performance time was measured before and at various times after treatment.
Irwin Test
The test was performed according to the method described by Irwin [1966].
Spontaneous Activity Meter (Animex)
Locomotor activity in mice was quantified using an Animex activity meter Type S (LKB, Farad, Sweden) set to maximum sensitivity. Every movement of mice, which were placed on the top of the Animex activity meter, pro-duced a signal due to variation in inductance and capac-ity of the apparatus resonance circuit. Signals were then automatically converted to numbers. On the day of the experiment the mice were treated and the cage,
contain-Fig.1. Chemical structure of ET-142 (2-(4-chlorophenoxy)propionic acid, endo 8-methyl-8-azabicyclo[3.2.1]oct-3-yl ester) and SS-20 (2-[4-chlorophenylthio]propionic acid, endo 8-methyl-8-azabicyclo[3.2.1]oct-3-yl ester).
ing five mice, was put on the measuring platform. Activity counts were made every 15 min for 45 min starting imme-diately after injection of the drug. Because of the arbitrary scale adopted to quantify movements, drug-treated mice were always compared with saline-treated ones.
Isolated Guinea Pig Ileum
The myenteric plexus longitudinal muscle was pre-pared according to Paton and Vizi [1969]. The strip was suspended in a 12.5 thermoregulated bath (36–37°C) and, after stabilization, the strip was stimulated electrically (0.1 Hz, 0.5 ms; double threshold voltage). The Krebs-Henseleit solution, bubbled with 95% O2 and CO2, had
the following composition (mM): NaCl 118.0, KCl 4.7, MgSO4.7H2O 1.2, CaCl2 2.5, KH2PO4 1.2, NaHCO3 25.0,
and glucose 11.0.
Acetylcholinesterase Activity
Acetylcholinesterase (AChE) activity was assayed according to Ellman et al. (1961), using 0.5 mM acetylth-iocholine iodide as substrate. The inhibitory effects of ET-142 and SS-20 were tested at various concentrations on a purified preparation of AChE from the electric eel.
Intracerebroventricular Injection Technique
Intracerebroventricular (icv) administration was performed under ether anesthesia using isotonic saline as solvent, according to the method described by Haley and McCormick [1957]. Briefly, during anesthesia mice were grasped firmly by the loose skin behind the head. A hypodermic needle of 0.4 mm external diameter attached to a 10 µl syringe was inserted perpendicularly through the skull at a depth of no more than 2 mm into the brain of the mouse, where 5 µl was then administered. The injection site was 1.5 mm from either side of the midline on a line drawn through to the anterior base of the ears. To ascertain that the drugs were administered exactly into the cerebral ventricle, some mice were icv injected with 5 µl of diluted 1:10 Indian ink and their brains examined macroscopically after sectioning.
Reagents and Drugs
The following drugs were used: ET-142 and SS-20 were prepared according to Gualtieri et al. [1994]; hemicholinium-3 hydrobromide (HC-3), pirenzepine dihydrochloride, naloxone hydrochloride (RBI, Natick, MA); morphine hydrochloride (U.S.L. 10/D, Florence); dicyclomine dihydrochloride (Lepetit); clomipramine (Anafranil), CGP 35348 (Novartis, Summit, NJ); ox-otremorine (Fluka, Buchs, Switzerland); atropine sulphate, atropine methylbromide, physostigmine emisulphate (Sigma, St. Louis, MO); ketorolac tro-metamine (Lixidol; Farmitalia Carlo Erba); diphenhy-dramine hydrochloride (De Angeli). Other chemicals
were of the highest quality commercially available. All drugs were dissolved in isotonic (NaCl 0.9%) saline solu-tion. Drug concentrations were prepared in such a way that the necessary dose could be administered in a vol-ume of 10 ml kg–1 by intraperitoneal (ip) and subcutane-ous (sc) route.
Statistical Analysis
Results are given as the mean ± S.E.M.; analysis of variance (ANOVA), followed by Fisher’s PLSD proce-dure for post hoc comparison, was used to verify the sig-nificance between two means. P values of less than 0.05
Fig. 2. Dose response curves of ET-142 (A) and SS-20 (B) administered sc in the mouse hot-plate test. The doses are expressed as mg kg–1 sc.
Vertical lines show s.e.m. ^P < 0.05; *P < 0.01 in comparison with saline controls. Each point represents the mean of at least 10 mice.
were considered significant. Data were analyzed with StatView for the Macintosh computer program (1992).
RESULTS
Antinociceptive Activity of ET-142 and SS-20
ET-142 (10–50 mg kg–1 sc; Fig. 2, panel A) and SS-20 (30–50 mg kg–1 sc; Fig. 2, panel B) produced a dose-de-pendent increase in the pain threshold in the mouse hot-plate test. A similar antinociceptive profile was obtained after icv of ET-142 (10–30 µg per mouse; Fig. 3, panel A) and SS-20 (5–30 µg per mouse; Fig. 3, panel B) adminis-tration. The antinociceptive effect of both compounds
peaked 15 min after injection and then slowly diminished. The maximum antinociceptive effects of ET-142 and SS-20 were greater than that produced by morphine (8 mg kg–1 sc), clomipramine (25 mg kg–1 sc), ketorolac (175 mg kg–1 sc), and diphenhydramine (20 mg kg–1 sc), used as rerence drugs (Fig. 4). Figure 5 illustrates the analgesic ef-fect of ET-142 and SS-20 in the mouse acetic acid abdominal constriction test where they increased the pain threshold at the dose of 10 and 20 mg kg–1 sc.
ET-142 and SS-20 were able to produce an increase in the pain threshold not only in mice but also in rats. In the paw pressure test, ET-142 and SS-20 administered ip at the dose of 20–40 mg kg–1 reached a maximum antinociception 15 min after injection and then slowly diminished (Table 1).
Antagonism of ET-142 and SS-20-Induced Antinociception
In the mouse hot-plate test, the antinociceptive ef-fects of ET-142 (40 mg kg–1 sc) and SS-20 (40 mg kg–1 sc) were not antagonized by the opiate antagonist naloxone (1 mg kg–1 ip), the peripherally acute cholinergic antagonist atropine methylbromide (5.5 mg kg–1 ip) or the GABAB
an-tagonist CGP-35348 (100 mg kg–1 ip) (Table 2). Conversely, atropine (5 mg kg–1 ip), dicyclomine (10 mg kg–1 sc),
Fig. 3. Dose response curves of ET-142 (A) and SS-20 (B) administered icv in the mouse hot-plate test. The doses are expressed as µg per mouse icv. Vertical lines show s.e.m. *P < 0.01 in comparison with saline con-trols. Each point represents the mean of at least 15 mice.
Fig. 4. Maximum antinociceptive effect of both ET-142 and SS-20 in comparison with morphine, clomipramine, ketorolac, and diphenhy-dramine evaluated in mouse hot-plate test. The nociceptive responses were recorded 15 min after administration of clomipramine, ketorolac, and diphenhydramine and 30 min after morphine injection. Each col-umn represents the mean of at least 10 mice. Vertical lines show s.e.m.
pirenzepine (0.1 µg per mouse icv), and hemicholinium-3 (1 µg per mouse icv) were able to completely prevent ET-142 and SS-20 antinociception in the mouse hot-plate and abdominal constriction tests (Table 2, Fig. 5). All antago-nists were injected 15 min before ET-142 and SS-20, with the exception of CGP 35348 and HC-3 which were injected 5 min and 5 h respectively before the two compounds.
Evaluation of the ET-142 and SS-20 Effect on Gross Behavior and Motor Coordination
ET-142 and SS-20, unlike oxotremorine and phys-ostigmine, increased the pain threshold without causing the typical cholinergic symptomatology (Table 3). Fur-thermore, both compounds elicited their antinociceptive effects without changing motor coordination, evaluated
Fig. 5. Dose-response curves of both ET-142 (A) and SS-20 (B) adminis-tered sc and antagonism exerted by dicyclomine (10 mg kg–1 i.p.) and
hemicholium-3 (1 µg per mouse i.c.v.) on antinociception induced by both compounds administered at the dose of 20 mg kg–1 ip in the mouse
abdominal constriction test induced by acetic 0.6% acid. The
nocicep-tive responses were recorded 15 min after ET-142 and SS-20 administra-tion. Dicyclomine and hemicholinium-3 were injected 5 min and 5 h respectively before ET-142 and SS-20. Vertical lines show s.e.m. *P < 0.01 in comparison with saline controls. °P < 0.01 in comparison with ET-142 or SS-20. Numbers inside the columns indicate the number of mice.
TABLE 1. Antinociceptive Effect of ET-142 and SS-20 in the Rat Paw-Pressure Test
Paw-pressure (g)
Treatment Dose Before After treatment
ip per kg–1 treatment 15 min 30 min 45 min
Saline 60.2 ± 3.8 63.7 ± 4.2 60.5 ± 3.4 64.2 ± 4.6 (10) (10) (10) (10) ET-142 20 mg 58.5 ± 3.8 90.6 ± 4.2** 78.4 ± 4.4* 64.8 ± 6.0 (5) (5) (5) (5) ET-142 40 mg 60.4 ± 4.0 134.6 ± 6.8** 123.6 ± 4.5** 84.6 ± 6.4* (7) (7) (7) (7) SS-20 20 mg 56.4 ± 3.9 93.4 ± 5.2** 64.6 ± 5.0 59.5 ± 4.3 (5) (5) (5) (5) SS-20 40 mg 60.6 ± 4.2 121.6 ± 5.5** 98.6 ± 5.2** 74.2 ± 4.0 (6) (6) (6) (6)
*P < 0.05; **P < 0.01 in comparison with controls. The number of rats is shown in parentheses.
TABLE 2. Effects of Atropine, Pirenzepine, Hemicholinium-3 (HC-3), Atropine Methylbromide, Naloxone, and CGP-35348 on Antinociception Induced by ET-142 (40 mg/kg–1 sc) and SS-20 (40 mg kg–1 sc) in the Mouse Hot-plate Test
Licking latency (sec)
Before After treatment
Pretreatment Treatment No. pretreatment 15 min 30 min 45 min
Saline Saline 36 14.4 ± 0.6 14.1 ± 1.1 13.7 ± 1.1 14.0 ± 0.3 10 ml kg–1 ip Saline Saline 16 14.1 ± 0.9 15.2 ± 1.1 14.3 ± 0.8 14.3 ± 0.5 5 µl icv Saline ET-142 20 14.2 ± 0.4 36.9 ± 2.41 25.3 ± 2.11 17.9 ± 0.8 ip or icv SS-20 22 14.5 ± 0.8 33.6 ± 2.11 29.8 ± 1.91 22.1 ± 1.31 Atropine Saline 10 14.2 ± 0.8 13.8 ± 1.2 14.4 ± 1.6 14.8 ± 1.8 5 mg kg–1 ET-142 10 14.3 ± 0.7 17.8 ± 2.53 17.1 ± 2.13 14.6 ± 1.7 ip SS-20 10 13.9 ± 0.9 18.1 ± 2.23 17.9 ± 1.83 15.2 ± 2.23 Pirenzepine Saline 10 14.3 ± 1.0 15.1 ± 1.6 15.3 ± 1.9 14.5 ± 1.7 0.1 µg mouse ET-142 12 14.0 ± 0.7 18.2 ± 2.53 14.7 ± 2.03 14.3 ± 1.4 icv SS-20 11 13.6 ± 0.9 16.9 ± 2.13 17.7 ± 1.43 13.8 ± 1.83 HC-3 Saline 10 15.1 ± 0.7 14.1 ± 1.5 15.2 ± 1.8 13.9 ± 1.7 1 µg mouse ET-142 12 13.3 ± 1.1 17.6 ± 2.33 17.5 ± 2.43 14.6 ± 1.9 icv SS-20 12 14.5 ± 0.8 16.8 ± 2.53 18.3 ± 2.53 16.7 ± 1.53 Atropine CH3Br Saline 10 14.5 ± 0.8 13.6 ± 1.6 15.7 ± 1.3 14.2 ± 1.1 5.5 mg kg–1 ET-142 11 13.5 ± 0.9 35.5 ± 2.21 26.4 ± 1.91 19.3 ± 1.92 ip SS-20 8 15.1 ± 0.7 33.9 ± 2.11 25.2 ± 2.31 18.9 ± 1.6 Naloxone Saline 10 14.5 ± 0.8 13.5 ± 1.2 13.4 ± 1.3 14.6 ± 1.4 1 mg kg–1 ET-142 9 13.8 ± 1.1 34.7 ± 1.81 27.4 ± 261 18.6 ± 2.1 ip SS-20 10 14.5 ± 1.1 31.7 ± 2.41 26.2 ± 1.81 20.3 ± 1.72 CGP 35348 Saline 10 13.0 ± 0.9 11.7 ± 1.42 13.5 ± 1.6 14.2 ± 1.3 100 kg–1 ET-142 10 13.9 ± 1.0 30.6 ± 2.01 26.8 ± 2.11 20.4 ± 1.72 ip SS-20 9 14.6 ± 1.1 32.1 ± 2.31 23.7 ± 2.11 19.7 ± 1.52 1P < 0.01.
2P < 0.05 in comparison with saline-saline. 3P < 0.01 vs. saline-ET-142/SS-20-treated mice.
by using the rotarod test (Table 3). The rotarod perfor-mance of mice treated with ET-142 and SS-20 at the dose of 50 mg kg–1 sc was not impaired in comparison with controls (Table 3). On the contrary, ET-142 and SS-20 administered at the dose of 60 mg kg–1 sc significantly
impaired rotarod performance (Table 3).
In Vitro Functional Studies
As shown in Figure 6, ET-142 and SS-20 added to the organ bath at concentrations ranging from 10–12–10–9
M potentiated the contractions evoked by electrical stimulation. The potentiation was no longer observed when the concentration of ET-142 and SS-20 in the
me-TABLE 3. Effect of ET-142 and SS-20 in Comparison With Oxotremorine and Physostigmine in: a) the Irwin test; b) Rotarod Test
Abdominal Spontaneous
a) Tremors Salivation Lacrimation Diarrhea tone motility
Saline 0 0 0 0 4 4 sc ET-142 0 0 0 0 4 4 50 mg kg–1 sc SS-20 0 0 0 0 4 4 50 mg kg–1 sc Oxotremorine 4 4 + + 0 2 0.1 mg kg–1 sc Physostigmine 2 6 + + 2 0 0.2 mg kg–1 sc
Tremors: absent = 0 maximum score = 8
Salivation: absent = 0 maximum score = 8
Lacrimation: absent = 0 present +
Diarrhea: absent = 0 present +
Abdominal tone: flaccid abdomen = 0 normal = 4 abdomen board-like = 8
Spontaneous motility: absent = 0 normal = 4 maximum score = 8
Each value represents the mean of five mice. Spontaneous motility was evaluated by Animex test.
b) Endurance time on rotarod(s)
Dose Before After treatment
mg kg–1 sc N treatment 15 min 30 min 45 min
Saline 12 97.6 ± 5.6 99.6 ± 6.8 102.4 ± 5.2 96.8 ± 8.3 ET-142 50 10 97.7 ± 5.8 95.6 ± 5.8 90.6 ± 4.2 101.5 ± 7.6 ET-142 60 10 103.2 ± 7.2 81.32 ± 5.4* 85.1 ± 7.2* 97.4 ± 7.7 SS-20 50 12 94.6 ± 6.8 98.1 ± 7.3 91.7 ± 5.4 94.9 ± 5.1 SS-20 60 8 97.5 ± 7.8 84.3 ± 7.2* 97.6 ± 6.2 104.2 ± 5.5 Oxotremorine 0.1 11 106.2 ± 8.2 76.5 ± 7.3* 63.6 ± 9.6** 64.4 ± 8.7** Physostigmine 0.2 9 93.4 ± 5.7 61.4 ± 6.8** 54.5 ± 8.1** 52.3 ± 8.8** *P < 0.05.
**P < 0.01 in comparison with saline controls. The number of mice is shown in parentheses.
Fig. 6. Dose-response curves of both ET-142 (closed symbols) and SS-20 (open symbols) on electrically (0.1 Hz; 0.5 ms; double-threshold voltage)-evoked contractions of guinea pig ileum myenteric plexus lon-gitudinal muscle strip expressed as percentage variation of contractions. Each point represents the mean of at least four experiments and vertical lines give s.e.m.
Figure 6.
dium was raised to 10–8 M. ET-142 and SS-20 began to
inhibit both types of evoked contractions at 10–6 M.
Finally, ET-142 and SS-20 had a weak antiacetyl-cholinesterase activity, with an IC50 value on electrical
eel acetylcholinesterase of 3 × 10–4 M (data not shown).
DISCUSSION
ET-142 and SS-20 were able to induce antino-ciception in mice regardless of which noxious stimulus was used: thermal (hot-plate test), chemical (abdominal constriction test), and mechanical (paw pressure). ET-142 and SS-20 antinociception was obtained without produc-ing any visible modification in animal gross behavior.
Moreover, motor coordination on the rotarod test was completely intact in mice treated with ET-142 and SS-20. The analgesic effect of the two compounds was also compared with that induced by well-known analgesic drugs such as morphine, clomipramine, ketorolac, and diphenhydramine at the highest doses that did not im-pair rotarod performance. By comparing the maximum antinociceptive activity, the enhancement of the pain threshold produced by ET-142 and SS-20 was greater than all the reference drugs.
ET-142 and SS-20 exerted their antinociceptive ef-fect by acting centrally. It was possible to enhance the pain threshold by injecting directly into the cerebral ven-tricles doses (30 µg per mouse) of ET-142 and SS-20 which were 1,000 times lower than those needed parenterally. Furthermore, a central cholinergic mecha-nism of action for ET-142 and SS-20 was hypothesized since their antinociception was prevented by the musca-rinic antagonist, atropine, the selective M1-antagonists,
dicyclomine and pirenzepine, and the ACh depletor, HC-3. Taking into account that HC-3 and pirenzepine were able to antagonize ET-142 and SS-20 antinociception af-ter icv injection and that atropine methylbromide admin-istered ip did not prevent ET-142 and SS-20 enhancement of the pain threshold, this supports the hypothesis that the analgesic site of action of the investigated compounds is localized in the central nervous system (CNS). A pre-synaptic mechanism facilitating cholinergic transmission is potentially involved in ET-142 and SS-20 anti-nociception as revealed by the antagonism of HC-3. A postsynaptic mechanism of action can potentially be ruled out since, as reported by Bartolini et al. [1987, 1992], HC-3 was not able to antagonize antinociception induced by agonists of postsynaptic muscarinic receptors such as oxotremorine, McN-A-343, and AF-102B.
The in vitro experiments supported the hypothesis that ET-142 and SS-20 amplify cholinergic neurotrans-mission since, ranging from 10–12–10–9 M, they increased presynaptically induced (electrical-evoked) contractions of longitudinal muscle of guinea pig ileum. ET-142 and SS-20 are also endowed with very low anticholinesterase activity, as demonstrated by the in vitro evaluation of their IC50 value (IC50 = 3 × 10
–4
M). It is possible that ET-142 and SS-20 are able to amplify cholinergic neurotrans-mission through a presynaptic mechanism and that this effect is in turn potentiated by their low cholinesterase inhibitory activity. However, we cannot exclude that other mechanisms able to potentiate the endogenous cholergic system may be involved in the antinociception in-duced by ET-142 and SS-20.
Opioid and GABAergic neurotransmitter systems are not involved in ET-142 and SS-20 antinociception since the opioid antagonist naloxone and the GABAB
an-tagonist CGP-35348 were unable to prevent the effect of
ET-142 and SS-20. The doses and administration sched-ules of the above-mentioned drugs were ideal for pre-venting antinociception induced by morphine [Ghelardini et al., 1992] and the GABAB agonist baclofen [Malcangio
et al., 1991].
In conclusion, our results indicate that ET-142 and SS-20 are able to produce dose-dependent antino-ciception in mice and rats by potentiating endogenous cholinergic activity and without impairing motor coordi-nation or spontaneous motility. Furthermore, both com-pounds show a very similar pharmacological profile, indicating that the substitution of a heteroatom (oxygen with sulphur) does not modify the antinociceptive activ-ity of ET-142 and SS-20.
ACKNOWLEDGMENT
The authors thank Ciba Geigy for the gift of CGP-35348.
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